Post-crystallization Improvement of RNA Crystal Diffraction Quality

Abstract

The crystallization and structural determination of large RNAs and their complexes remain major bottlenecks in the mechanistic analysis of cellular and viral RNAs. Here, we describe a protocol that combines post-crystallization dehydration and ion replacement that dramatically improved the diffraction quality of crystals of a large gene-regulatory tRNA–mRNA complex. Through this method, the resolution limit of X-ray data extended from 8.5 to 3.2 Å, enabling structure determination. Although this protocol was developed for a particular RNA complex, the general importance of solvent and counterions in nucleic acid structure may render it generally useful for crystallographic analysis of other RNAs.

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Acknowledgements

We thank the staff at beamlines 5.0.1 and 5.0.2 of the ALS and ID-24-C and ID-24-E of APS, in particular, K. Perry and K.R. Rajashankar of the Northeastern Collaborative Access Team (NE-CAT) of the APS for support in data collection and processing, G. Piszczek (National Heart, Lung and Blood Institute, NHLBI), R. Levine and D.-Y. Lee (NHLBI) for assistance with biophysical and mass spectrometric characterization, and N. Baird, T. Hamma, C. Jones, M. Lau, A. Roll-Mecak, and K. Warner for discussions. This work is partly based on research conducted at the ALS on the Berkeley Center for Structural Biology beamlines and at the APS on the NE-CAT beamlines (supported by National Institute of General Medical Sciences grant P41GM103403). Use of ALS and APS was supported by the US Department of Energy. This work was supported in part by the intramural program of the NHLBI, NIH.